Colostrum and Passive Immunity
Colostrum is a lactation product produced by the mother after birth (parturition) of mammals such as humans and cows. Typically, the term colostrum milk is used to designate the mammal milk produced for about 5 to 7 days after parturition. Colostrum milk contains an elevated concentration of immunoglobulins compared to normal milk (50-100 gram/L in colostrum compared to 0.5-1.0 gram/L in normal milk). For a range of mammals including cows and pigs the survival of the newborn mammal is critically dependent on the ingestion and successful absorption of immunoglobulins during the first 24 hours after parturition. These mammals are born without circulating antibodies in the blood and need to obtain them by so-called passive immunisation at birth. During the first few hours after parturition the intestine of the newborn is able to absorb the immunoglobulins and transport these to the blood path. An efficient passive immunisation is achieved if the newborn is able to obtain a serum concentration of circulating immunoglobulins in the range of 10-20 gram/L.
Required Colostrum Intake
The amount of colostrum to feed a calf depends on several factors—including the amount of antibody (or immunoglobulin) in the colostrum, the body weight of the calf, the age of the calf at first feeding, and several other factors. In order to calculate the amount (or mass) of IgG that a calf needs, several assumptions may be made, based on existing research data (see list below). The goal is for the calf to obtain a minimum of 10 grams of IgG per liter of serum. A calf's plasma volume at 24 hours of age is approximately 9% of its body weight. To achieve 10 g/L, a newborn calf that weighs 40 kg (about 88 lbs.) must consume 36 grams of IgG from colostrum or a supplement by 24 hours of age. However, IgG is not absorbed with 100% efficiency. Research data suggest the efficiency is closer to 35% (the other 65-70% equilibrates with other body pools or is not absorbed at all). So, to achieve 10 g/L, the calf must consume 103 grams of IgG (36 grams/35%) by 24 hours. If a margin of safety is included in the calculations (achieving a plasma IgG concentration of 15 grams of IgG per liter), the calf needs to consume 154 grams of IgG.
Estimated Colostrum Required by a 40 kg Calf to Achieve Minimum Plasma IgG Concentration.
Calf body weight40kgPlasma volume (9% of BW)3.6litersMinimum Plasma concentration10g/LApparent efficiency of absorption35%Required IgG intake103gramsColostral concentration50g/LRequired colostrum amount to feed2.1LPassive Immunity and Infectious Diseases Acquired by the New Born from Colostrum or Milk
A key component of the survival and health of calves is colostrum feeding in the first 24 hours of life. Veterinarians and farmers have known for more than one hundred years the importance of colostrum feeding in maintaining the health of young animals, including calves, foals, kids, lambs, pigs, cats and dogs. Research has shown that the absorption of IgG in the first 24 hours of life determines the degree of acquisition of passive immunity and subsequent resistance to disease. The USDA has estimated that nearly 11% of dairy calves in USA die prior to weaning; most of the mortality can be attributed to inadequate colostral IgG intake. The USDA also estimated that 41% of dairy heifer calves have failure of passive transfer at 24 hours of age. The amount of IgG absorbed by the calf is determined by many factors, including the concentration of IgG in colostrum, feeding practices of the farm and metabolic state of the animal. Colostrum is widely variable in IgG concentration, and unfortunately, it is difficult to measure colostral IgG concentration.
The presence of contaminants in colostrum and milk adds a degree of risk to neonatal feeding. Colostrum and milk is recognized as a vector for transmission of a number of disease causing organisms, including the widespread Mycobacterium avium subsp. paratuberculosis (in short: Mycobacterium paratuberculosis) causing Johne's disease in cattle. Farms with significant Johne's infestation often have inadequate supplies of colostrum to feed to their newborn calves. Consequently, farmers are forced to rely on feeding milk replacers and using large amounts of antibiotics to keep the animal alive until its own active immune system can protect it.
Mycobacterium paratuberculosis is the agent of a chronic, fatal, granulomatous enterocolitis in ruminants also called paratuberculosis or Johne's disease. Johne's disease is a chronic, debilitating intestinal disorder in cattle characterized by diarrhea, reduced feed intake, weight loss and death.
In cattle, the course of the disease is as follows. Calves acquire the infection in the first months of life through oral uptake of colostrum, milk or feces of infected cows. They either successfully clear the infection or become subclinically infected for life. The subclinically infected animals shed the bacteria in their feces intermittently or continuously from an age of approximately two years onward. After an incubation period of four to five years, a proportion of the subclinically infected animals develops an incurable progressive form of protein-losing enteropathy with chronic diarrhea that is ultimately fatal.
A dairy survey conducted by the American National Animal Health Monitoring System suggests that between 20 and 40% of dairy herds in the United States are affected by Johne's disease and it is expected that this figure will continue to increase unless producers implement management regimes that will help control the spread of this disease within their herds. Economic losses are estimated to be $200/infected cow/year and are the result of animal culling, reduced milk production, poor reproductive performance, and reduced carcass value. Johne's disease has become a high priority disease in the cattle industry. The economic impact of this disease on the US dairy industry was estimated to be over $200 million per year in 1996 and is growing each year with the continued spread of this disease. In addition, M. paratuberculosis has been implicated as a causative factor in Crohn's disease, a chronic inflammatory bowel disease of human beings, which has served as a further impetus to control this disease in our national cattle.
The neonatal calf is a target for infection with Mycobacterium paratuberculosis, the causative agent of Johne's disease. Calves become infected via exposure to the bacterium through contaminated feces, bedding, colostrum, and milk. Shedding of viable M. paratuberculosis has been documented in the colostrum and milk of infected dams. However, symptoms of disease do not usually present themselves until the animals reach 3 to 5 years of age or even older. During this time the animal is infected and may be shedding the organism in its feces without showing any clinical signs of disease. In addition to reduced milk production by these animals, they also present a potential infective threat to the rest of the herd. Johne's disease is difficult to manage and control on-farm.
Johne's disease is known to affect cattle, sheep, llamas, camels, goats, farmed deer, bison, and other domestic and wild ruminants. It may be the cause of some wasting diseases in horses and swine. Also, chickens can be successfully infected with it. The disease can also be transmitted to laboratory animals in a laboratory setting. It may also be an agent of the Crohn's disease in humans. There is conflicting information about the zoonotic risks of the disease, but interaction with diseased animals should be done with caution. Johne's is found worldwide.
Johne's disease control programs on dairy farms require testing of cows. Calves born to cows with positive or presumptive positive tests must be removed from the dam to avoid fecal contamination and must be fed colostrum from test negative cows. Unfortunately, many herds are unable to collect sufficient test negative colostrum. In addition, because tests are notoriously unreliable with a test sensitivity that is less than 50%, there is a significant risk that test negative cattle will also shed the Johne's causative organism in colostrum and/or feces.
Most susceptible to infection are animals in their first month of life, although clinical disease becomes apparent only several years later. The main way of infection is the fecaloral route, but since excretion of M. paratuberculosis has been demonstrated in colostrum as well as in milk of infected cows, there are strong concerns about feeding such mycobacteria loaded colostrum or normal milk to highly susceptible neonate calves.
Mycobacteria are notoriously resistant to physical and chemical factors but M. paratuberculosis seems to be among the most resistant of mycobacteria.
The conventional way of reducing the content of microorganisms in milk is pasteurization and ultra-heat treatment i.e. the milk is exposed to heat for a short period of time. However, the heat treatment does not only destroy the microorganisms present in the milk, but also denatures essential biologically active proteins such as the immunoglobulins.
Several studies have investigated the effect of pasteurisation. The thermal tolerance of M. paratuberculosis, specifically the capacity to survive pasteurization, is the subject of considerable interest. Some published reports suggest that M. paratuberculosis can survive standard commercial pasteurization while others suggest it cannot. Thermal tolerance curves indicate that M. paratuberculosis is comparable in heat resistance to M. avium and far more heat resistant than Listeria, another facultative intracellular bacterium that is found in raw milk. It has been suggested that mycobacteria in milk or colostrum may be killed by pasteurisation at high temperature and thus eliminate the risk of infection of neonates and newborn animals through this route. However, high temperature treatment of immunoglobulin solutions including colostrum has been shown to denature or lead to uncontrolled aggregation and inactivate the biological activity of the immunoglobulins and it seems impossible to perform an efficient and complete elimination of the mycobacterium from colostrum or milk under conditions that does not at the same time influence the biological activity of the fragile immunoglobulin molecules present in colostrum. In addition, pasteurizing colostrum at conventional temperatures can result in unacceptable feeding characteristics.
Other important pathogens typically found in colostrum and raw milk include various viruses and bacteria including Salmonella spp., Listeria monocytogenes, Escherichia coli, Campylobacter spp., Streptococcus spp., Staphylococcus spp
A method of reducing the bioburden of colostrum by centrifugation has been described in WO97/16977. However, an effective reduction of bacteria requires such a high force of gravitation that proteins might precipitate together with other particles present in a protein rich solution.
Other methods of reducing microbial contaminants in milk are gamma radiation (U.S. Pat. No. 4,784,850) and treatment with β-propiolactone (U.S. Pat. No. 3,911,108). Also, these methods tend to denature proteins to some extent. Sterile filtration is still another method of removing microbes from milk (U.S. Pat. No. 5,256,437; U.S. Pat. No. 5,683,733. Filtration does not usually substantially affect the proteins, but the filters rapidly foul when the solution to be filtered is complex, comprise lipids, colloidal and/or insoluble substances. This is especially a problem with protein rich colostrum, which easily clogs the filters. The problem with clogged filters has previously been solved by partially or completely removing casein from the colostrum, and/or by diluting the colostrum before filtration. Casein can be removed by either acid or enzymatic precipitation, and centrifugation to obtain whey (U.S. Pat. No. 4,644,056 and GB 1,573,995). U.S. Pat. No. 5,670,196 discloses a method of microfiltrering colostrum, whereby defatted colostrum is first acidified to precipitate casein, which is removed by centrifugation, and then the whey is filtered through a charged depth filter to reduce the microorganism content. U.S. Pat. No. 5,707,678 is directed to a similar method, where casein is removed, after which the acidified whey is first ultrafiltered and then microfiltered. The main drawback of these methods is that large amounts of valuable antibodies and other proteins tend to precipitate together with the casein. In addition the removal of casein is a laborious, time consuming and expensive process.
U.S. Pat. No. 5,147,548 discloses a method of sterile-filtering colostrum without previously removing the casein. The optionally defatted colostrum is acidified to a pH of less than 3.5. The casein precipitates at a pH of 5 to 4, but it returns into solution as the pH continues to drop. The acidic solution was found to differ so extensively from the original colostrum that it could be sterile filtered either as such or after neutralizing it back to its original pH. The filter used is a depth filter or a membrane filter. In a preferred embodiment the colostrum is diluted into a sodium chloride solution prior to acidifying. However, also this method has drawbacks. The immunoglobulins are easily inactivated at low pH. Further, the casein precipitation is not fully reversible resulting also in protein loss, and the dilution of the colostrum increases process time and expenses.
WO05006861 deals with disinfecting blood and blood fractions using disinfectant dyes and more specifically with a simple method to destroy a large number of pathogens in human blood prior to transfusion. There is no mentioning of using disinfecting dyes for inactivation of pathogens in colostrum, milk or whey.
EP01094846 is related to the medical field of haematology and, more particularly, for an improved method and device for the removal of disinfectant dyes such as methylene blue from blood, blood fractions or other perishable liquids to which the dyes have been added for disinfecting purposes. The disclosure is silent with regard to the possible application of the method for removal of disinfectant dyes from colostrum, milk or whey.
Thus, there is a need for new methods of inactivating or removing infectious agents, such as mycobacteria, in milk and colostrum; milk and colostrum replacers and milk and colostrum supplements without concomitant loss of protective antibody activity and other important qualities.
Accordingly, there is an interest in providing a milk and/or colostrum; milk and/or colostrum replacer and milk and/or colostrum supplement, which has been treated to inactivate or remove infectious agents, such as mycobacteria, substantially without loss of protective antibody activity and other important qualities.
Further, there is an interest in providing a method for use of such inactivated products integrated with the general handling of newborn mammals particularly farm production animals such as newborn calves, goats kids, lambs and piglets in order to minimise the risk of infection.
Antimicrobial Compositions (Also Described Herein as Conjugates)
Structurally, antibodies are composed of one or more units, or monomers, each typically portrayed as resembling a Y shape. A monomer contains four polypeptides—two identical copies of a polypeptide known as the heavy (H) chain, and two identical copies of a polypeptide called the light (L) chain. The two heavy chain polypeptides are approximately 440 amino acids long having a molecular weight of about 55,000 daltons each. The two light chains are approximately 220 amino acids long and each having a molecular weight of about 25,000 daltons.
One light chain associates with one heavy chain, and any one antibody molecule will have only one type of light chain and one type of heavy chain. The amino-terminal variable region of a light chain associates with the amino-terminal variable region of one heavy chain to form an antigen binding site. The carboxy-terminal regions of the two heavy chains fold together to make the Fc domain.
The four polypeptide chains of the resulting immunoglobulin molecule are held together by disulfide bridges and non-covalent bonds.
Antibodies are divided into classes—IgG, IgM, IgA, IgE and IgD—on the basis of the type of heavy chain polypeptide they contain. There are only two types of light chain proteins, kappa and lambda. The classes also vary in the number of monomers that join to form a complete antibody molecule. For example, IgM antibodies have five monomers, each with two antigen binding sites, yielding ten identical antigen binding sites for each molecule.
IgM is thus referred to as decavalent. IgG, IgE, and IgD typically consist of a single monomeric unit and thus are bivalent, and IgA may consist of one, two or more monomers. An antibody's intrinsic affinity is a measure of the strength of its binding to an epitope. In principle, it represents the binding by one antigen binding region, i.e., one-half of a monomer's total binding sites. Avidity, on the other hand, is a measure of the overall stability of the complex between antibody and antigen. Avidity is effected by the intrinsic affinity of the antibody for the epitope, the valency of the antibody and antigen, and the geometric arrangement of the interacting components.
Multivalent interactions may allow low affinity antibodies to bind antigens tightly and can greatly stabilize immune complexes. Thus, antibodies of high avidity may possess increased therapeutic or diagnostic value compared to similar antibodies of low affinity and low avidity
In humans, secretion of IgA by the mucosal immune system accounts for 70% of the body's total Immunoglobulin production. The secretory IgA (S-IgA having four antigen binding sites) in the mucociliary blanket is believed to afford protection to mucosal surfaces by neutralizing or otherwise preventing the attachment of viruses, bacteria, and toxins to the mucosal epithelium. In contrast IgG is the predominant immunoglobulin found in the blood.
Mammals, in particular new born calves, suffering from a depressed or an insufficient immune system, or mammals that may be expected to be suffering from a depressed or insufficient immune system may be treated by passive immunisation to control microbial infections. Passive immunisation of humans and animals by intravenous or intramuscular administration of immunoglobulin G is well described in the art for a broad and steadily increasing range of indications.
US application no.: 2005-0191289 discloses non-covalent complexes of immunoglobulins for passive immunisation by systemic absorption through the enteral (oral) and/or transmucosal route. The purpose of the non-covalent complexing with e.g. chitosan is to protect the immunoglobulin against gastrointestinal proteases and acidic environment.
Most disease-causing organisms first enter the body through mucosal surfaces, which is not surprising since the surface area of human mucosae (including the gastrointestinal, genito-urinary, and respiratory tracts) exceeds that of skin by a factor of 200. Most of the antibody that is synthesized daily is secreted by epithelial cells at mucosal surfaces, where it forms a first line of defense against infection. The vast majority of all antibody-secreting plasma cells are located in the intestinal Iamina propria, a layer of cells underlying the epithelial cell layer. These plasma cells produce more IgA than all other immunoglobulin isotypes combined (in humans, 40-60 mg/kg/day). The IgA is secreted as polymeric IgA (SIgA), which is comprised mostly of IgA dimers (4 heavy chains and 4 light chains), linked together by a protein called the joining, or J chain.
Complexes of SIgA and antigen are removed from the body by physical mechanisms, such as peristalsis and mucociliary transport in the gastrointestinal and respiratory tracts.
In contrast to vaccines, passive immunization can deliver protective levels of antibodies directly to the susceptible mucosal site where most infections begin. Passive protection by transfer of antibody from maternal origin represents in many species including humans an efficacious and specific mechanism to prevent mucosal infection in newborns lacking a fully matured immune system. This takes place by ingestion of breast milk containing SIgA antibodies induced by natural exposure of the maternal immune system to environmental microbes.
The natural protection offered by passive immunization has already provided the physicians with the opportunity to envisage treatments against mucosal infectious agents with antibody-enriched milk, serum or egg yolk preparations (examples in Table 1).
TABLE 1Examples of Immunotherapy by Administration of Antibody Onto Mucosal SurfacesType ofInfectiousTargetIg DeliveredInfectionAgentTargeted AntigenOrganismIgGRespiratory tractRespiratoryF glycoprotein, GPMouseinfectionsyncytial virus84IgG2aDental cariesStreptococcusSA I/IIHumanmutansIgARespiratory tractSendai virusHN proteinMouseinfectionSIgAStreptococcalGroup AM proteinMouseinfectionStreptococciIgA, IgGNecrotizing—MultipleHumanenterocolitisIgG, IgM,MeningoccocalE. coli K1b-polysaccharideRabbitIgAinfectionIgAFluPR8 InfluenzaHemagglutininMousevirusIgA, IgGRespiratory tractSendai virusMultipleMouseinfectionIgAPseudomembranousClostridiumMultipleHumancolitisdifficileIgAGastritis, duodenalHelicobacterMultipleMouseulcersfelisIgADiarrheaCampylobacterMultipleHumanjejuniIgA, SIgACholeraVibrio choleraeLipopolysaccharideMouseFab (LI)Respiratory tractRespiratoryF glycoproteinMouseinfectionsyncytial virusIgACholeraVibrio choleraeLipopolysaccharideMouseIgYDiarrhea,RotavirusMultipleCalfgastroenteritisIgARespiratory tractRespiratoryF glycoproteinMouseinfectionsyncytial virusIgAGastritis, duodenalHelicobacterUreaseMouseulcersfelisIgADysenteryShigella flexneriLipopolysaccharideMouseIgARespiratory tractRespiratoryF glycoproteinMonkeyinfectionsyncytial virusIgGRespiratory tractRespiratoryF glycoproteinCalfinfectionsyncytial virusIgASexually transmittedChlamydiaMajor OMPMousediseasetrachomatisIgG, IgARhinitis—MultipleHumanIgG,Dental cariesStreptococcusSA I/IIHumanSIgA/GmutansIgYPseudomembranousClostridiumToxin A, Toxin BHamstercolitisdifficileIgADiarrheaCryptosporidiumP23MouseparvumIgG, IgARespiratory tractRespiratoryF glycoproteinMouseinfectionsyncytial virusIgG1Respiratory tractRespiratoryF glycoproteinRatinfectionsyncytial virusSIgADysenteryShigella FlexnerLipopolysaccharideMouse
SIgA has a number of advantages over the monomeric IgG for passive immunization at skin and mucosal surfaces. First, the presence of four antigen binding sites confers an increase in avidity. Second, SIgA is more resistant than IgG or monomeric IgA to the proteolytic enzymes found in the gastrointestinal tract.
For passive immunization at skin and mucosal surfaces to afford protection against infection, it is likely that a few milligrams/kg will be required daily during some susceptible period, whereas systemic clearance of an existing infection might require much higher amounts.
Even though SIgA is secreted at high rates at the mucosal surfaces there is no easy route to isolate and produce concentrates of SigA for use in large scale passive immunisation programs.
SIgA has been produced in transgenic plants and by other genetically modified organisms. However, these attempts to source this type of antibody are still very expensive to realise commercially and for each new antibody specificity there is a need for a corresponding transgenic or other genetically modified organism. In addition, even though the antibody produced may be identical to the antibodies corresponding to the species of application, there is a strong demand to remove any contaminating host antigens that may evoke allergic or other adverse reactions.
WO9106305 discloses the preparation of oligomeric monoclonal immunoglobulin G with high avidity for antigens. The oligomeric antibodies are produced by the use of genetically engineered organisms and are typically no more than tetra or hexylent with respect to antigen binding sites. Again, this approach to prepare high avidity antibodies is very expensive to realise commercially and still for each new antibody specificity there is a need for a corresponding genetically modified organism. In addition, even though the antibody produced may be identical to the antibodies corresponding to the target species of final therapeutic application, there is a strong demand to remove any contaminating host antigens that may evoke allergic or other adverse reactions.
IgG and monomeric IgA, however is available for isolation on a large scale and at low cost from e.g. human and animal blood plasma and milk. Particularly large amounts of IgG are available in the bovine milk industry. One litre of milk or whey from the cheese industry may provide 300-1000 mg IgG and is therefore available in sufficient quantities to support an industrial production of products for passive immunisation. Likewise animal plasma from slaughterhouses is abundant and contains a high concentration of IgG and IgA. Human plasma is systematically collected from blood transfusion centers and is available commercially for the isolation of immunoglobulins as well. The antibodies present in all of these raw materials has polyvalent antigen binding specificity reflecting the environment and exposure to diverse antigens of the host and pooling of blood or milk from several hosts will create a very broad antibody specificity thus eliminating the need for individual production of monoclonal antibody preparations. In certain instances it may even be advantageous to vaccinate the hosts (animals or humans) in order to provoke an immune response against a particular antigen or microorganism—or a cocktail of antigens or microorganisms.
Another group of substances exhibiting antimicrobial activity is a non-immunoglobulin substance, such as Lactoferrin.
Mammary fluids, colostrum and milk, deliver nature's first host defense systems upon birth, and these essential liquids are critical for survival of the neonate. The identification and characterization of substances, such as anti-infectious proteins, were among the early scientific discoveries and this group of proteins has long been recognized for promoting health benefits in both newborns and adults. Among the more widely studied are the immunoglobulins, lactoperoxidase, lysozyme, and lactoferrin.
“Antimicrobial peptide” (“AmP”), as used herein, refers to oligopeptides, polypeptides, or peptidomimetics that kill (i.e., bacteriocidal) or inhibit the growth of (i.e., bacteriostatic) microorganisms including bacteria, yeast, fungi, mycoplasma, viruses or virus infected cells, and/or protozoa. In some instances, AmPs have been reported to have anticancer activity. Generally antimicrobial peptides are cationic molecules with spatially separated hydrophobic and charged regions. Exemplary antimicrobial peptides include linear peptides that form an α-helical structure in membranes or peptides that form β-sheet structures optionally stabilized with disulfide bridges in membranes. Representative antimicrobial peptides include, but are not limited to, cathelicidins, defensisn, dermeidin, and more specifically magainin 2, protegrin, protegrin-1, melittin, 11-37, dermaseptin 01, cecropin, caerin, ovispirin, and alamethicin. Naturally occurring antimicrobial peptides include peptides from vertebrates and non-vertebrates, including plants, humans, fungi, microbes, and insects.
Lactoferrin is one of the principal proteins responsible for providing protection to infant mammals before their immune systems begin to function. It is a minor protein in cow's milk (100-300 mg/L) and is extracted from skim milk or whey through protein separation. As an iron-binding glycoprotein of the transferrin family, Lactoferrin is found in high concentrations in mother's milk. It is used throughout the US, Europe and Asia as a nutritional supplement or as an additive to infant formula.
Apart from milk, lactoferrin is generally produced and released in the body in the digestive, respiratory and reproductive systems through saliva, tears, nasal secretions, etc. Lactoferrin is also produced by a special group of white blood cells known as neutrophils.
The presence of lactoferrin in these biological fluids points to lactoferrin's primary role as the principal gatekeeper of the non-specific defense system against invading pathogens and other disease causing agents.
Most of the biological activity of lactoferrin is believed to be related to its excellent iron binding properties, but many non-iron binding related effects have been described as well.
With respect to its role as a primary pathogen defense protein, research supports the following important functionalities:                Antimicrobial activity, including Gram positive and Gram negative bacteria, yeast and fungi        Anti-viral activity, including cytomegalovirus, influenza, HIV and rotavirus        Anti-oxidant activity, protects the white blood cells against free iron catalyzed oxidation reactions        Immune system enhancement, controls the body's immune response during infection and inflammation.        
The antimicrobial activity of lactoferrin has several working mechanisms:                Lactoferrin binds iron which is an essential element for bacterial growth. Bacterial cells become iron deprived and stop growing.        Lactoferrin binds to bacteria and, as a consequence, the microbial cell membrane loses its integrity and the bacteria is killed.        Lactoferrin stimulates phagocytosis or microbial destruction by macrophages and monocytes.        Lactoferrin eliminates and prevents bacteria from forming essential attachment structures making them incapable of colonizing and multiplying.        
Many scientific studies have demonstrated these potent antimicrobial effects. Recent studies also show an important potentiating effect of lactoferrin with antibiotics and antifungal agents. In vivo results from animal studies are available as well. Bovine lactoferrin was shown to have a bacteriostatic effect in mice infected with either Enteric pathogens or Clostridia. In humans the duration and severity of enteric infections decreased when patients were treated orally with lactoferrin. Bacterial infections in rainbow trout could be reduced by oral administration of bovine Lactoferrin.
Recently a significant amount of attention has been given to the antiviral activity of lactoferrin. The mechanism of action appears to be the inhibition of the absorption process of the virus particle to the mammalian host cell, either through binding to the host cell or to the virus itself. Published scientific studies have clearly demonstrated the protective effects of lactoferrin against HIV and cytomegalovirus, Herpes simplex type 1 and 2, hepatitis C, influenza and rotavirus. Interestingly, the first in vivo animal studies support the findings from the in vitro work. When mice were pretreated with bovine lactoferrin and challenged with mouse cytomegalovirus it did not result in the typical 100% mortality. The mechanism behind this protective effect is related to the stimulation of natural killer cell activity (phagocytosis).
Neutrophils, monocytes and macrophages are cells of the immune system that kill invading pathogens by oxidation reactions. Free iron is often present in areas of inflammation or infection. Oxidation reactions are accelerated by the catalytic effect of iron on free radical production. Lactoferrin binds the free iron with extremely high affinity and thus functions as a powerful local antioxidant protecting the immune cells against the free radicals produced during the inflammatory response. Although only the neutrophils produce and deliver lactoferrin, monocytes and macrophages have lactoferrin receptors on their cell surfaces.
One of the most potent stimulants of cytokine activity, (compounds produced by immune cells during infection and inflammation to coordinate the defense against pathogens), is the endotoxin LPS. This microbial membrane derived lipopolysaccharide (LPS) is bound and neutralized by lactoferrin and down regulates the immune response before it can get out of control as is the case with autoimmune disease. Both in vitro and in vivo studies have shown these protective and stimulatory effects of Lactoferrin.
Lactoferrin in a polymerised (complexed) form has been introduced commercially for the treatment of meat surfaces (e.g. carcasses) in the food industry. The product called Activin from aLF Ventures is apparently based on a non-covalent interaction between Lactoferrin and naturally occurring polysaccharides. The Activin product is claimed to be one thousand times more effective in inhibiting attachment of pathogenic bacteria than normal monomeric Lactoferrin.
U.S. Pat. No. 6,172,040 disclose a method for treating products, such as meat products, with immobilized lactoferrin to reduce microbial contamination. The lactoferrin is immobilized on a naturally occurring substrate, preferably a galactose-rich polysaccharide, via the N-terminal region. In some embodiments, the lactoferrin is applied as an aqueous solution containing a mixture of the immobilized lactoferrin and native lactoferrin, and a buffer system that includes a physiologically acceptable acid, such as citric acid, a physiologically acceptable base, such as sodium bicarbonate, and a physiologically acceptable salt, such as sodium chloride. There is no mentioning or teaching of a covalent bonding of Lactoferrin to the substrate and all examples given in the disclosure are based on non-covalent—and therefore reversible—interactions.
However, the stability of the complex between Lactoferrin and a substrate such as a polysaccharide can be highly critical for the efficiency of the Lactoferrin. Any reversibility in the complex towards the monomeric Lactoferrin will lead to a diminished effect. This will be particularly relevant in cases where a Lactoferrin complex may be exposed to extreme pH values and/or an ionic strength high enough to dissociate the non-covalent interactions keeping the complex intact (such as passage through the stomach and the gastrointestinal tract or exposure to other mucosal surfaces).
Accordingly, what is needed in the art is a Lactoferrin complex having a covalent structure and a fixed high molecular weight providing enhanced anti-microbial activity of Lactoferrin under challenging pH and salt conditions.
Thus, it is of interest to provide a germ free composition comprising colostrum, a composition comprising synthetically multimerised immunoglobulins, having antimicrobial activity and high avidity and yet which avoid many of the difficulties and costs associated with the isolation of similar substances on a large scale or preparation of genetically engineered organisms giving narrow antigen binding specificities. Furthermore, it is of interest to provide a more clinically effective and commercially viable product for passive immunisation of skin, skin lesions, surgical wounds and mucosal surfaces.